SU553938A3 - The method of obtaining the tryptophan and its derivatives - Google Patents

Description

(54) THE METHOD OF OBTAINING L-TRIPTOFAN AND ITS DERIVATIVES

ycnoMtax endlessly adding a nutrient substrate with a separate, uninterrupted outward compression to the system of the corresponding longitudinal derivative.

The strain used is characterized by a high conversion rate of indole to L tryptophan. The strain can be grown on a medium of a certain composition, and for its growth, in addition to carbon. Dov and thiamine (vitamin Bg), do not require any other organic matter.

The used strain Candicfa humicola has a partially active pathway for the metabolism of tryptophan, including its decomposition, catalyzed by tryptophan-pyrrolase.

However, by changing the culture conditions and the composition of the nutrient medium, it is possible to suppress the decomposition process of tryptophan. In order to stimulate the formation of mutants incapable of decomposing tryptophan, niacin can be added to the medium in a concentrate of 0.05-0.5 g / l.

The indole fed in the required amount into the KjTibtural medium penetrates the cell wall and is converted by the intracellular: i enzyme systems into tryptophan, which in a significant part is again removed from the cell into the culture fluid. Relatively ezkie concentration of free indole can reduce the growth rate of cells.

However, the conversion of indole to tryptophan occurs very quickly and the addition of, for example, 0.5% (w / v) of indole relative to the weight of the substrate in the fermentation system will only lead to an insignificant content of free indole in the fermentation medium.

In this regard, it is very important to determine the content of indole in the fermenter, calculated on the substrate containing the cells of the microorganism, since a very large amount of free indole is inside the cells.

A feature of the proposed method, which seems to have the greatest effect on the conversion rate of indole to tryptophan and on the degradation of tryptophan through kinurenin, is the presence in the culture fluid of a certain concentration of biologically digestible iron, for example, in the form of ferrous acid.

The concentration of iron (Fe) in the culture fluid must be lower than the amount of iron that causes the decomposition of tryptophan, but higher than the amount at which the growth of the culture is disturbed and the continuous cultivation becomes thus impossible.

The rate of decomposition of tryptophan is influenced by the amount of solution of oxygen in the fermentation medium, since tryptophan-pyrrolase is an enzyme containing a heme group, and the decomposition of tryptophane is thus an oxidative process.

With a low level of aeration in the culture

liquids can be maintained at a relatively high concentration, with a high level of aeration, the iron content should be low. These observations are valid for Candida strain.

wild type humicola.

However, in the process of uninterrupted fermentation for a long time, mutants may occur that do not decompose tryptophan. This is facilitated by the conditions of cultivation: the composition of the medium (especially the presence of niacin in the medium), the indol feed, etc., In this CJQ4ae, the concentration of iron in the medium will not be decisive for the decomposition of tryptophan and it can be maintained at a much higher level .. Therefore, molasses can be used as a carbon source, usually a high iron content.

In order not to create a high concentration of residual indole in the medium, which suppresses

strain growth, it is necessary to periodically analyze.

The oxygen level of the fermentation medium increases significantly with an increase in the concentration of residual indole in the medium, as a result of which, under these conditions, the culture changes from aerobic to anaerobic type of metabolism. This parameter can be used to control the rate of iron citrate or degree of aeration.

Q The number of cells also serves as a criterion, according to

which can be judged on the course of the process, the changes in this case occur so slowly that it is almost impossible to use them to control the fermentation process.

The residual indole is determined by gas chromatography in samples containing cells (the determination of the indole in the fermentation fluid that is free of the cells does not give the correct

Q results, since the indole is rapidly absorbed by the cells).

Too high a content of residual indole can be used as a signal to reduce the feed rate of the indole into the system and / or increase the iron supply and / or to increase the hydration.

The yield of tryptophs is controlled by continuous analysis on a Sephadex column using Wickord and subsequent analysis of tryptophan fractions in the ultraviolet. This method can be used to determine some metabolites of tryptophan and, mainly, kinurenins, the acid. The ability of the strain used to synthesize tryptophan is not limited by inhibition

j according to the type of feedback. The culture of Candida humicola withstands up to 15 g / l of tryptophan in the medium without any change in the rate of its synthesis.

The factor limiting the yield of tryptophan according to the described method is the ability of the microorganism to synthesize sarin. The consumption of indole by the strain increases sharply with the addition of serine to the enzymatic medium.

Since the series is an expensive substance, the effect of glycine additives on tryptophan was studied. Glycine has the same effect on tryptophan yield as the series, but this effect does not appear immediately, but several hours after the addition of glycine to the medium.

With the addition of glycine in the amount of 1-10 g / l, the rate of introduction of indole into the fermentation medium can be significantly increased.

The method of ion exchange is used to isolate and purify the resulting tryptophan from the culture fluid freed from the cells. After passing the culture liquid through the resin column, the resin is rinsed with distilled water. The elution of tryptophan is carried out with a solution containing 50% ethanol and 50% 1 M ammonia solution, using the counter-current method.

The resulting tryptophan solution is neutralized with hydrochloric acid, and crystalline tryptophan is obtained, the yield of which is about 95%. The product has 100% biological activity according to the standard test with S. fecal is.

The proposed method is illustrated by examples that do not limit it.

Example. Low speed of indole addition and high iron content.

Fermenter. A Himap type apparatus (Chemap 3F 0014) with a mechanical defoamer, a revolutions number of 2,000 rpm, a cy6cTpaiTa addition rate of 200 Nm / h, a volume of 4.8 l, a dilution rate: 4.1%. Temperature 26 ° C. Substrate. Indole, g

p - Aminobenzoic acid (PABK), g 01 Yeast extract, g25

Solution A, ml Solution B, ml Solution C, ml Glucose (sterilized separately), kg

To 10

Distilled water, l pH 5.5

Solution A: 3% CaCl 2 H2O. Solution B; 10% KHjRO4.

Solution C: 5% MgSO4 2H20, 0.125% iron citrate, 0.02% ZnSO4 7HjO, and 10% NaCl.

Aeration 80 l / h l medium.

The fermenter is seeded with 50 ml of a one-day cultured Candida humicola grown on a rocking chair. The pH of the culture process is maintained at about 6.5 with ammonia.

The culture is grown for 24 hours, after

What can be done with strata and indole. The feed rate of the schodol is set at 0.14% (w / v). In relation to the feed rate of the substrate.

When the content of iron citrate is 24 mg / l, the rate of consumption of indole is high, and no residual indole is observed. However, under these conditions, tryptophan is practically not produced, probably as a result of the fact that the resulting tryptophan immediately decomposes to kinurenic acid and other metabolites.

PRI mme R 2. Low content of indole and low iron content.

Conditions are the same as in the example. The difference in the composition of the nutrient medium;

NH4CI, g50

0.5

Niadin, g

J10

Iron citrate, mg

50 Thiamine mg

ten

Solution K, ml

100

Solution A, ml

200 Solution B1, ml

200 Solution Br, ml

200 Solution C, ml

Sucrose, kg1

Distilled water, ldo 10

Solution A: 0.75% CaCl2 -21120

PactBOp BI: 8% KHj P04.2.56% K2 HPO4

ftcTBOp B2: 10% KH2PO4

Solution С ..: 7.88% MgSO4 7Н2О, 0.11% MnSO4 НЗО, 0.02% ZnS04 7Н20 and 10% NaCl.

Solution K: 0.068 g ammonium molybdate, 0.34 g CuSO4. Sowing, pH and cultivation are the same as in Example 1. The rate of addition of the indole

0.265% with respect to the rate of substitution. rata. The yield of tryptophan during the enzymatic process is 3.6 g / l day (the efficiency of conversion of indole to tryptophan is 78%). No decomposition of tryptophan is observed.

PRI me R 3. High content of indole

and periodic addition of limonocleslo iron.

The fermentation conditions are the same as in Example 1. The nutrient medium is as in Example 2. Sowing, pH and

the cultivation is the same as in Example 1. The rate of addition of the indole to the fermentation medium is 0.53% (w / v) of the rate of introduction of the substrate. The yield of tryptophan during the fermentation process averages 6.8 g / l day (process efficiency 74%). Iron limonokis